Usually, the presence of biologically active agents such as bacteria in a patient's body fluid, and especially in blood, is determined using blood culture vials. A small quantity of blood is injected through an enclosing rubber septum into a sterile vial containing a culture medium. The vial is typically incubated at 37.degree. C. and monitored for bacterial growth.
Common visual inspection involves monitoring the turbidity or observing eventual color changes of the liquid suspension. Known instrumented methods detect changes in the carbon dioxide content of the culture bottles, which is a metabolic byproduct of the bacterial growth. Monitoring the carbon dioxide content can be accomplished by methods well established in the art, such as radiochemical (e.g., BACTEC.RTM., Becton-Dickinson, Franklin Lakes, N.J., USA), infrared absorption at a carbon dioxide spectral line (e.g., NR-BACTEC.RTM., Becton-Dickinson, Franklin Lakes, N.J., USA), or pressure/vacuum measurement such as those disclosed in U.S. Pat. No. 4,152,213--Ahnell. However, all these methods require invasive procedures which result in the well-known problem of cross-contamination within the vial. For purposes of this application, the term invasive implies that the confines of the sample container must be entered in order to determine if bacteria are present, e.g., a probe is inserted into a sealed vial. In the first two methods mentioned above, the headspace gas must be removed for analysis. In the case of vacuum/pressure measurement, while pressure is measured in a closed vial, any temperature change within the vial headspace also generates a pressure change that is not related to biological activity.
Therefore, an additional headspace temperature measurement is required in order to distinguish between biological and temperature-generated pressure effects. Non-invasive headspace temperature monitoring, however, represents a difficult problem, and no practical solutions are at hand. Additionally, some microorganisms can produce high pressure values while others produce relatively low or negligible ones. Thus, any pressure sensors used must be sensitive enough to allow detection of small changes in pressure while also being capable of safely measuring high pressure values. These two requirements are often mutually exclusive depending on the type of pressure sensor technology used. Thus far none of the pressure measurement systems known in the prior art permit the rapid and reliable detection of a wide variety of bacteria.
In order to circumvent the problems of cross-contamination, a non-invasive infrared microorganism detection instrument has been proposed in which special vials with infrared-transmitting windows are utilized. See U.S. Pat. No. 4,889,992--Hoberman. These vials, however, are relatively expensive. There is also an instrument in which glass vials are transferred to an infrared spectrometer by an automated manipulator arm and measured through the glass vial. This instrument is known as the BIO AGROS and is distributed by Diagnostics Pasteur, France. The disadvantage of this system is that, due to the high infrared absorption of glass, small changes in the glass wall thickness generate large errors in the measured headspace gas absorption. These problems can be partly reduced by utilizing high-quality glass vials, but this measure results in relatively high vial cost.
Recently, non-invasive methods have been developed involving chemical sensors disposed inside the vial. These sensors respond to changes in the carbon dioxide concentration by changing their color or by changing their fluorescence intensity. See, e.g., Thorpe, et al. "BacT/Alert: an Automated Colorimetric Microbial Detection System" J. Clin. Microb., July 1990, pp. 1608-12 and U.S. Pat. No. 4,945,060. These techniques are based on light intensity measurements and require spectral filtering in the excitation and/or emission signals. This means that errors can occur if any of the light source, the photodetector, the filters, or the sensor show aging effects over time which would vary the intensity response.
The disadvantage of such intensity-based methods can be overcome by utilizing a modulated excitation signal in combination with fluorescent sensors that change their fluorescent decay time with changing carbon dioxide concentration. In such a device, light intensity measurement is replaced with time measurement, and intensity changes and the related variations in sensor sensitivity therefore have no impact upon its operation. However, current fluorescent decay time sensors require high-brightness, short-wavelength light sources (550 nm or shorter) that are intensity-modulated at very high frequencies (typically about 100 MHz). Thus, for example, such a system might use a 5-mW green HeNe laser (543.5 nm), externally modulated by means of an acousto-optic light modulator, the operation of which is understood by those of ordinary skill. However, it will be realized that such a laser/modulator combination is rather expensive, requiring that the samples be moved to the laser, instead of having one light source at each sample. Such an instrument would therefore by necessity have a complicated mechanism for effecting the transportation of the individual samples to the light source and the time interval between successive measurements for each sample would be relatively long. It appears unlikely that high-brightness short-wavelength semiconductor diode lasers will be developed in the near future. Thus, even such an improved system would suffer serious practical shortcomings.
Thus, there remains an unmet need to provide a system for detecting microorganisms in samples that are sealed in vials that can tolerate the errors caused by the defects found in inexpensive glass vials. It is therefore an object of the present invention to overcome the limitations of the prior art described above by providing methods and apparatus for detecting biological activities in a specimen such as blood that are non-invasive and that do not require chemical sensors or any other additives within the specimen container. It is a further object of this invention to disclose a system that does not require high-brightness, short-wavelength light sources while also providing methods and apparatus that are safe against eventual extreme high pressure values while not being sensitive to headspace temperature changes. Another object of the present invention is to provide a system that is simple and inexpensive, so that each vial can be monitored continuously, thus allowing the construction of diagnostic instruments containing a plurality of stationary vials.